Total RNA was recovered and purified using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was synthesized using a QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). First-strand cDNA was prepared from 1 μg of total RNA. The samples were subjected to real-time PCR using a SYBR Green master mix on a Rotor-Gene Q 2plex System (Qiagen, Hilden, Germany). The gene expression levels were normalized to those of GAPDH. Relative gene expression changes were calculated using the 2-delta CT method and reported as a fold change over the control samples. The sequence of primers was as follows: CCL26, (forward) 5′-AAC TCC GAA ACA ATT GTG ACT CAG CTG-3′ and (reverse) 5′-GTA ACT CTG GGA GGA AAC ACC CTC TCC-3′; GAPDH, (forward) 5′-TGT GTC CGT CGT GGA TCT GA-3′ and (reverse) 5′-CCT GCT TCA CCA CCT TCT TGA-3′. The primers used in this experiment were purchased from Bioneer (Daejeon, Korea).
Rotor gene q 2plex system
Manufactured by Qiagen
Sourced in Germany
The Rotor-Gene Q 2plex system is a real-time PCR cycler designed for multiplex gene expression analysis. It features dual-channel detection and can accommodate up to 72 samples. The system includes the Rotor-Gene Q instrument and associated software.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green master mix, by Qiagen (10 mentions)
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Quantitect reverse transcription kit, by Qiagen (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Reverse transcriptase premix, by Elpis Biotech (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Primescript first strand cdna synthesis kit, by Takara Bio (3 mentions)
Fam zen ibfq probes, by Integrated DNA Technologies (3 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Retroscript kit, by Thermo Fisher Scientific (2 mentions)
Primerquest program, by Integrated DNA Technologies (2 mentions)
Hex zen ibfq probes, by Integrated DNA Technologies (2 mentions)
Rneasy protect mini kit, by Qiagen (2 mentions)
Prime taq premix, by GeNet Bio (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Lung dissociation kit, by Miltenyi Biotec (1 mentions)
Rneasy ffpe kit, by Qiagen (1 mentions)
Nanodrop nd 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
24 protocols using rotor gene q 2plex system
1
Quantifying CCL26 Gene Expression
2
Quantitative Analysis of Inflammatory Markers in Allergic Rhinitis
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Lung tissues were isolated from allergic rhinitis mice model and homogenized for mRNA extraction using lung dissociation kit (Miltenyi biotec, Bergisch Gladbach, Germany). Total RNA was recovered and purified using an RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. cDNA was synthesized using a QuantiTect Reverse Transcription Kit (Qiagen, Germany). First-strand cDNA was prepared from 1 μg of total RNA. The samples were subjected to real-time PCR using SYBR Green master mix on a Rotor-Gene Q 2plex System (Qiagen, Germany). The gene expression levels were normalized to those of β-actin. Relative gene expression changes were calculated using the 2-delta CT method and reported as fold change over the control samples. The sequence of primers was as follows: IL-8, (forward) 5′-CGG CAA TGA AGC TTC TGT AT-3′ and (reverse) 5′-CCT TGA AAC TCT TTG CCT CA-3′; IL-1β, (forward) 5′-ACCTGGGCTGTCCTGATGAGAG-3′ and (reverse) 5′-GTT GAT GTG CTG CTG CGA GAT-3′; TNF-α, (forward) 5′-TCT TCT CAT TCC TGC TTG TGG-3′ and (reverse) 5′-GGT CTG GGG CAT AGA ACT GA-3′; IL-6, (forward) 5′-TGG GAC TGA TGC TGG TGA CAA C-3′ and (reverse) 5′-AGC CTC CGA CTT GTG AAG TGG T-3′; β-actin, (forward) 5′-GCT CAG TAA CAG TCC GCC TAG A-3′ and (reverse) 5′-TGT CCA CCT TCC AGC AGA TGT-3′. The primers used in this experiment were purchased from Bioneer (Daejeon, Korea).
3
Quantification of SHP gene expression
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Total RNA was extracted from 10-µm slices of FFPE samples from potentially eligible patients (n=12) using a Qiagen RNeasy® FFPE kit (Qiagen, Inc.) following the manufacturer's protocol. RNA quantitation and purity were measured using a NanoDrop™ ND-8000 spectrophotometer (Thermo Fisher Scientific, Inc.).
cDNA was synthesized by RT using SuperScript™ II Reverse Transcriptase (cat. no. 18064; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCRs were carried out using Rotor-Gene SYBR-Green PCR kits (cat. no. 204074; Qiagen, Inc.) in a Rotor-Gene Q 2plex system (cat. no. 9001620; Qiagen, Inc.). The samples were amplified for 40 cycles as follows: 95°C for 10 sec and 60°C for 30 sec. To analyze the qPCR data, relative quantification was performed using the 2−ΔΔCq method with human GAPDH as the internal control gene; data are expressed as relative fold-changes (32 (link)). The sequences of human SHP and GAPDH primers were as follows: SHP forward, 5′-TCCTCTTCAACCCCGATGTG-3′ and reverse, 5′-CAGGGTTCCAGGACTTCACAC-3′: GAPDH forward, 5′-TCGGAGTCAACGGATTTGGT-3′ and reverse, 5′-TTCCCGTTCTCAGCCTTGAC-3′.
cDNA was synthesized by RT using SuperScript™ II Reverse Transcriptase (cat. no. 18064; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCRs were carried out using Rotor-Gene SYBR-Green PCR kits (cat. no. 204074; Qiagen, Inc.) in a Rotor-Gene Q 2plex system (cat. no. 9001620; Qiagen, Inc.). The samples were amplified for 40 cycles as follows: 95°C for 10 sec and 60°C for 30 sec. To analyze the qPCR data, relative quantification was performed using the 2−ΔΔCq method with human GAPDH as the internal control gene; data are expressed as relative fold-changes (32 (link)). The sequences of human SHP and GAPDH primers were as follows: SHP forward, 5′-TCCTCTTCAACCCCGATGTG-3′ and reverse, 5′-CAGGGTTCCAGGACTTCACAC-3′: GAPDH forward, 5′-TCGGAGTCAACGGATTTGGT-3′ and reverse, 5′-TTCCCGTTCTCAGCCTTGAC-3′.
4
Isolating and Analyzing PBMCs from Venous Blood
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Human PBMCs were isolated from heparinized venous blood using Ficoll-Hypaque (Lymphoprep; Axis-shield, 1114544, Dundee, Scotland) following the manufacturer’s manual. Total RNA from PBMCs was extracted using QIAzol lysis reagent (Qiagen, 1023537, Hilden, Germany) and miRNeasy Mini Kits (Qiagen, 217004) according to the manufacturer’s instructions, followed by RNA quantitation and assessment using QIAxpert (Qiagen).
After RNA quantitation, complementary DNA (cDNA) from total RNA was synthesized using reverse transcription master premix (Elpis Biotech, EBT-1515, Daejeon, South Korea). For miRNA expression analysis, cDNA from total RNA was synthesized using miScript II RT Kits (Qiagen, 218161), as described previously17 (link). qRT-PCR was performed in the Rotor-Gene Q 2plex system (Qiagen, 9001620) using SYBR Green master mix (Qiagen, 204076) or miScript SYBR Green PCR Kit (Qiagen, 218073) and primers for the indicated genes. The primer sequences are shown in Supplementary Table1 . Relative expression levels of mRNA and miRNA were calculated with the 2−ΔΔCt method.
After RNA quantitation, complementary DNA (cDNA) from total RNA was synthesized using reverse transcription master premix (Elpis Biotech, EBT-1515, Daejeon, South Korea). For miRNA expression analysis, cDNA from total RNA was synthesized using miScript II RT Kits (Qiagen, 218161), as described previously17 (link). qRT-PCR was performed in the Rotor-Gene Q 2plex system (Qiagen, 9001620) using SYBR Green master mix (Qiagen, 204076) or miScript SYBR Green PCR Kit (Qiagen, 218073) and primers for the indicated genes. The primer sequences are shown in Supplementary Table
5
Quantifying Yeast Transcript Levels
6
Gene Expression Analysis by RT-qPCR
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mRNA was extracted and purified with the RNeasy Mini Kit (Qiagen, Venlo, the Netherlands), and cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen). Real time RT-PCR was performed in a Rotor-Gene Q 2plex System (Qiagen) as follows: 10 min at 95 °C, followed by 45 cycles of 30 s at 95 °C, and 30 s at 58–62 °C. Primer sequences are provided in Supplementary Information, Table S2 . Transcript levels were normalized to an internal control (GAPDH, HPRT, or β-actin).
7
Quantitative PCR of PBMC RNA
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Total RNA from PBMCs or MDMs was extracted using QIAzol lysis reagent (Qiagen, Hilden, Germany) and miRNeasy Mini Kits (Qiagen) according to the manufacturer's instructions, followed by RNA quantitation. cDNA from total RNA was synthesized using the reverse transcription master premix (ELPIS Biotech, Daejeon, Korea). RT-qPCR was performed in Rotor-Gene Q 2plex system (Qiagen) using SYBR green master mix (Qiagen) and primers for indicated genes. Primers used in this study are listed in Supplementary Table 1 . Data were analysed using 2ΔΔ threshold cycle method where GAPDH was used for normalization.
8
Profiling Epithelial-Mesenchymal Transition Markers
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The mRNA expression levels of PD-L1, PD-L2, E-cadherin, N-cadherin, Vimentin, and Snail1 were analysed using a Rotor-Gene Q 2plex System (Qiagen, Hilden, Germany) with FAM/ZEN/IBFQ probes (Integrated DNA Technologies, Inc., Coralville, IA, USA; DNA sequences not opened). Total RNA was extracted using the RNeasy Protect Mini kit (Qiagen), and cDNA was obtained using the PrimeScript first-strand cDNA Synthesis kit (Takara, Tokyo, Japan). All reactions were performed according to the manufacturer's instructions. We amplified 18S rRNA as an internal standard using HEX/ZEN/IBFQ probes (Integrated DNA Technologies, Inc.; DNA sequences not opened). Relative expression levels were calculated using the ΔΔCt method for qPCR (34 (link)), which presents the data as fold-differences in expression level relative to a calibrator sample; in this case, the mean expression of 3 experimental measurements of 18S rRNA in control cells or vehicle-treated cells.
9
Quantitative Real-Time PCR Analysis of Gene Expression
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For in vivo samples, lung tissue suspended in PBS was homogenized and centrifuged to retrieve cell pellets before maintenance at −80 °C. In vitro samples were harvested by removing the supernatants from cultured BMDMs. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, 15596026) and used for synthesis of complementary DNA (cDNA) with Reverse Transcriptase Premix (Elpisbio, EBT-1515). Next, qPCR was carried out using cDNA, primers, and SYBR Green Master Mix (Qiagen, 204074) following the manufacturer’s protocol. Reactions were conducted on a Rotor-Gene Q 2plex system (Qiagen) and qPCR data were analyzed using the delta-delta CT relative quantification method with the Rotor-Gene 6000 Series software. Data were expressed as relative fold changes compared to the expression of the control gene β-actin. The primers used are summarized in Supplementary Table S1 .
10
Quantitative RT-PCR analysis of regulatory genes
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RNA from spleens and mLNs was extracted and purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen). RT-PCR was performed in a Rotor-Gene Q 2plex System (Qiagen) as follows: 10 min at 95°C, followed by 45 cycles of 30 s at 95°C and 30 s at 62°C. The Assays-on-Demand kit (Qiagen) was used to analyze the expression of HPRT (housekeeping gene), Foxp3, CTLA-4, AhR, and Granzyme B (GranB). The sequences of the primers used are provided in Table 1 . HPRT was used to normalize mRNA expression. The data are shown as relative delta delta CT (ΔΔCT); the fold-induction of each gene was calculated as follows: ΔThreshold cycle (ΔCt) = (Ct of target mRNA) − (Ct of HPRT); ΔΔCt = (ΔCt of mRNA in target gene) − (ΔCt of mRNA in control gene); fold-induction = 2−ΔΔCt.
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